Process for actinol production from ketoisophorone

ABSTRACT

Disclosed is a process for producing actinol from ketoisophorone which comprises contacting ketoisophorone with a recombinant microorganism or cell-free extract thereof in a reaction mixture, wherein said recombinant microorganism is obtainable by transforming a host microorganism, e.g. selected from the group consisting of microorganisms of the genera  Saccharomyces, Zygosaccharomyces,  and  Candida,  such as commercially available baker&#39;s yeast,  Saccharomyces cerevisiae  ATCC7754,  Saccharomyces rouxii  ( Zygosaccharomyces rouxii ) HUT7191 (IFO 0494),  Saccharomyces delbrueckii  HUIT7116 ( Saccharomyces unisporus  IFO 0298),  Saccharomyces delbrueckii  ( Torulaspora delbrueckii ) HUT7102,  Saccharomyces willianus  HU7106,  Zygosaccharomyces bailii  ATCC11486,  Candida tropicalis  IFO 1403, and a mutant thereof, which is capable of reducing ketoisophorone to levodione with a levodione reductase gene, e.g. a levodione reductase gene derived from a microorganism belonging to the genus  Corynebacterium,  such as  C. aquaticum  AKU611 (FERM BP-6448) or a mutant thereof, and isolating the produced actinol from the reaction mixture.

The present invention relates to a one-step process for the microbialproduction of (4R, 6R)-4-hydroxy-2,2,6-trimethylcyclohexanone(hereinafter referred to as actinol) from2,6,6-trimethyl-2-cyclohexene-1,4-dione (hereinafter referred to asketoisophorone) and to recombinant organisms involved in such process.

Actinol is useful for the synthesis of carotenoids, such as zeaxanthin.EP 982,406 discloses an efficient microbial production of actinol whichcomprises contacting levodione with a microorganism which is selectedfrom the group consisting of microorganisms of the genera Cellulomonas,Corynebacterium, Planococcus, and Arthrobacter, and which is capable ofselective asymmetric reduction of levodione to actinol, and recoveringthe resulting actinol from the reaction mixture. EP 1,026,235 disclosesan enzyme, levodione reductase, that acts on levodione to produceactinol, which was isolated from Corynebacterium aquaticum AKU611 (FERMBP-6448). The genetic material such as an isolated DNA comprising anucleotide sequence coding for an enzyme having levodione reductaseactivity, a polypeptide encoded by such a DNA, recombinant organisms andthe like, are disclosed by EP 1,122,315.

EP 1,074,630 discloses an efficient microbial production of levodionewhich comprises contacting ketoisophorone with at least one kind ofyeast capable of converting ketoisophorone into levodione and selectedfrom the group of species consisting of Saccharomyces rouxii(Zygosaccharomyces rouxii), Saccharomyces delbrueckii (Saccharomycesunisporus, Torulaspora delbrueckii), Saccharomyces willianus,Zygosaccharomyces bailii, and Candida tropicalis. Isolation andcharacterization of an enzyme ketoisophorone reductase that acts onketoisophorone to produce levodione was recently reported.

Until now there was no biological one-step process for the directproduction of optically active actinol from ketoisophorone, whichrequires two sequential reactions from ketoisophorone to levodione, andlevodione to actinol.

The present invention is directed to a process for the production ofactinol from ketoisophorone with high optical purity by a single stepreaction using a recombinant micro-organism which is capable of reducingketoisophorone to levodione, and levodione to actinol simultaneously.

More particularly, the present invention provides a process forproducing actinol from ketoisophorone by contacting ketoisophorone witha recombinant microorganism or cell-free extract thereof which has bothketoisophorone-reducing activity and levodione-reducing activity becauseof introduction of ketoisophorone reductase gene or levodione reductasegene, or both, and isolating the resulted actinol from the reactionmixture.

In another aspect the present invention provides a process for producingactinol from ketoisophorone by contacting ketoisophorone with bothketoisophorone reductase, which is capable of catalyzing the conversionof ketoisophorone to levodione, and levodione reductase, which iscapable of catalyzing the conversion of levodione to actinol,simultaneously.

The present invention further provides a recombinant microorganism, thatis obtained by transforming a host microorganism which is capable ofreducing ketoisophorone to levodione with levodione reductase gene.

More particularly, the present invention provides a recombinantmicroorganism, that is obtained by transforming a host microorganismwhich is capable of reducing levodione to actinol with ketoisophoronereductase gene.

In still another aspect the present invention provides a recombinantmicroorganism which expresses both ketoisophorone reductase gene andlevodione reductase gene.

In the present invention, any recombinant microorganism which is capableof producing actinol from ketoisophorone can be used. In the presentinvention, any microorganism which is capable of producing levodionefrom ketoisophorone can be used as a host microorganism to obtain arecombinant microorganism which is capable of producing actinol fromketoisophorone by introducing levodione reductase gene in it. Saidmicroorganism can also be used as a donor strain for ketoisophoronereductase gene. A preferred microorganism is selected from the groupconsisting of microorganisms of the genera Saccharomyces,Zygosaccharomyces, and Candida. Examples include S. cerevisiae ATCC7754,S. rouxii (Z. rouxii) HUT7191 (IFO 0494), S. delbrueckii HUT7116 (S.unisporus IFO 0298), S. delbrueckii (Torulaspora delbrueckii) HUT7102,S. willianus HUT7106, Z. bailii ATCC11486, C. tropicalis IFO 1403,mutants thereof, and even commercially available baker's yeast(available from e.g. the Oriental Yeast Co., Ltd.). These yeasts aredisclosed in EP 1,074,630, and have been deposited with at least one ofthe depositaries American Type Culture Collection (ATCC), 10801University Boulevard Manassae, Va. 20100-2209, USA; HUT CultureCollection Room, Department of Fermentation Technology, HiroshimaUniversity; and Institute for Fermentation Osaka (IFO), 17-85Jusohonmachi 2-chome, Yodogawa-ku, Osaka, Japan. Each is available fromthe pertinent depositary to anyone upon request.

In the present invention, any microorganism which is capable ofproducing actinol from levodione can be used as a host microorganism toobtain a recombinant microorganism which is capable of producing actinolfrom ketoisophorone by introducing ketoisophorone reductase gene in it.Said microorganism can also be used as a donor strain for levodionereductase gene. A preferred microorganism is selected from the groupconsisting of microorganisms of the genera Cellulomonas,Corynebacterium, Planococcus, and Arthrobacter, including Cellulomonassp. AKU672 (FERM BP-6449), Corynebacterium aquaticum AKU610 (FERMBP-6447), C. aquaticum AKU611 (FERM BP-6448), P. okeanokoites AKU152(IFO 15880), A. sulfureus AKU635 (IFO 12678), and mutants thereof. Thesemicroorganisms are disclosed in EP 982,406. Cellulomonas sp. AKU672(FERM BP-6449), Corynebacterium aquaticum AKU610 (FERM BP-6447) and C.aquaticum AKU611 (FERM BP-6448) were deposited at the National Instituteof Advanced Industrial Science and Technology (AIST), Tsukuba Central 6,1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan, in the name of F.Hoffmann-La Roche AG of Grenzacherstrasse 124, CH-4070 Basle,Switzerland on Aug. 4, 1998, under Budapest Treaty. P. okeanokoitesAKU152 (IFO 15880) and A. sulfureus AKU635 (IFO 12678) were deposited atIFO under the deposit No. described above, and any one can publiclyobtain them from the depository. One of the most preferred strains is C.aquaticum AKU611.

In the present invention, any microorganism which is originally notcapable of producing levodione from ketoisophorone, nor actinol fromlevodione, can be used as a host micro-organism to obtain a recombinantmicroorganism which is capable of producing actinol from ketoisophoroneby introducing both genes encoding ketoisophorone reductase andlevodione reductase in it.

Either a growing or a resting cell culture or immobilized cells or acell-free extract, or the like, of said recombinant microrganism may beused for the production of actinol. A growing cell culture can beobtained by culturing a recombinant microorganism in a nutrient mediumcontaining saccharides such as glucose or sucrose, alcohols, such asethanol or glycerol, fatty acids, such as oleic acid and stearic acid oresters thereof, or oils, such as rapeseed oil or soybean oil, as carbonsources; ammonium sulfate, sodium nitrate, peptone, amino acids, cornsteep liquor, bran, yeast extract and the like, as nitrogen sources;magnesium sulfate, sodium chloride, calcium carbonate, potassiummonohydrogen phosphate, potassium dihydrogen phosphate, and the like, asinorganic salt sources; and malt extract, meat extract, and the like, asother nutrient sources.

Cultivation of the recombinant microorganism can be carried outaerobically or anaerobically at pH values from 4.0 to 9.0, at atemperature in the range of from 10 to 50° C. for 15 min to 72 hours,preferably, at pH values from 5.0 to 8.0, at a temperature in the rangeof from 20 to 40° C. for 30 min to 48 hours.

Using the growing cell culture thus obtained, a resting cell culture orimmobilized cell or cell-free extract may be prepared by any meansgenerally known in the art.

The concentration of ketoisophorone in a reaction mixture can varydepending on other reaction conditions, but, in general, may be between0.1 g/l and 300 g/l, preferably between 1 g/l and 30 g/l.

In the present invention, actinol can also be produced by contactingketoisophorone with ketoisophorone reductase and levodione reductaseconcomitantly.

One of the most preferred levodione reductases and a method for itspreparation is described in EP 1,026,235. This enzyme was characterizedby the following physicochemical properties:

1) The levodione reductase catalyzes regio- and stereoselectivereduction of levodione to actinol.

2) The relative molecular mass of the enzyme is estimated to be 142,000-155,000±10,000 Da, consisting of four homologous subunits having amolecular mass of 36,000±5,000 Da.

3) The optimum temperature is 15-20° C. at pH 7.0 and the optimum pH is7.5.

4) The enzyme requires NAD⁺ or NADH as a cofactor and is highlyactivated by mono-valent cations, such as K⁺, Na⁺, Cs⁺, Rb⁺, and NH₄ ⁺.

Levodione reductase catalyzes the reduction of levodione to actinol inthe presence of a co-factor according to the following formula:levodione+NADH

actinol+NAD

Ketoisophorone reductase and its genetic material such as an isolatedDNA comprising a nucleotide sequence coding for an enzyme havingketoisophorone reductase activity can be obtained from a microorganismwhich is capable of reducing ketoisophorone to levodione. A preferredmicroorganism is selected from the group consisting of microorganisms ofthe genera Saccharomyces, Zygosaccharomyces and Candida, including S.cerevisiae ATCC7754, S. rouxii (Z. rouxii) HUT7191 (IFO 0494), S.delbrueckii HUT7116 (S. unisporus IFO 0298), S. delbrueckii (Torulasporadelbrueckii) HUT7102, S. willianus HUT7106, Z. bailii ATCC11486, C.tropicalis IFO 1403, mutants thereof, and even commercially availablebaker's yeast (available from e.g. the Oriental Yeast Co., Ltd.). Thesemicroorganisms are disclosed in EP 1,074,630.

The enzyme reaction may, e.g., be performed as follows: The basalreaction mixture (total volume: 1 ml): 200 μl of 1 M potassium phosphatebuffer (pH 7.0), 40 μl of 8 mM NADH in 0.2 mM KOH, 200 μl ofketoisophorone solution, and 20-80 μl of the enzyme solution, and waterup to 1 ml, is incubated at pH values of from 4.0 to 9.0, at atemperature range of from 10 to 50° C. for 5 min to 48 hours, preferablyat pH values of from 5.0 to 8.0, at a temperature range of from 20 to40° C. for 15 min to 24 hours.

The concentration of ketoisophorone in a reaction mixture can varydepending on other reaction conditions, but, in general, may be between0.1 g/l and 300 g/l, preferably between 1 g/l and 30 g/l.

Actinol produced biologically or enzymatically in the reaction mixtureas described above may be extracted by an organic solvent such as ethylacetate, n-hexane, toluene, or n-butyl to recover the actinol into theorganic solvent layer. The extract is analyzed by known methods such asgas chromatography, high performance liquid chromatography, thin layerchromatography or paper chromatography, or the like.

After the reaction, actinol in the reaction mixture may be recovered,for example, by extraction with a water-immiscible organic solvent whichreadily solubilizes actinol, such as ethyl acetate, n-hexane, toluene orn-butyl acetate. Further purification of actinol can be effected byconcentrating the extract to directly crystallize actinol or by thecombination of various kinds of chromatography, for example, thin layerchromatography, adsorption chromatography, ion-exchange chromatography,gel filtration chromatography or high performance liquid chromatography.

The following Examples further illustrate the present invention, butthese are not thereby limiting the scope of the invention.

EXAMPLE 1

Production of Levodione By Reducing Ketoisophorone Using S. cerevisiaeINVScI Resting Cells

S. cerevisiae INVScI was inoculated into YP-raffinose medium (5 ml in atest tube) containing 10 g/l of Bacto Yeast Extract, 20 g/l of BactoPeptone, and 20 g/l of raffinose, and cultivated at 30° C. for 20 hr. Aportion of the seed culture was inoculated into the same medium (10 mlin a test tube) and cultivated at 30° C. for 5 hr. Initial opticaldensity value for 600 nm wavelength of the cultivation was adjusted to0.2. After addition of 20% galactose solution (1 ml), cultivation wascontinued for 16 hr. The main culture broth of each test tube wascollected and harvested by centrifugation. The pellet of thecentrifugation was used as a resting cell of the followingketoisophorone-reducing reaction after wash with 0.1 M of KPB (pH 6.0).

The ketoisophorone-reducing reaction was done as follows. 0.11 g ofresting cells were suspended into 1 ml of reaction buffer, which is 0.1M of KPB (pH 6.0) containing 50 g/l of glucose and 5.0 g/l ofketoisophorone, and incubated at 30° C. in a test tube with gentlyshaking (200 rpm). The reaction solution was recovered by centrifugingthe reaction mixture and removing resting cell pellet. Resultingreaction solution was then extracted with equal volume of ethyl acetateand analyzed by gas chromatography [column: BGB-176 (BGB Analytik AG,Switzerland), instrument: GC-14A (SHIMADZU, Japan), carrier gas: He,column temperature: 1° C./min elevation from 100° C. to 150° C.,injector temperature: 200° C.]

The result of the reaction is described in Table 1. After 17.0 hrreaction, 2.8 g/l of levodione was produced from 5.0 g/l ofketoisophorone. Concentration of a major by-product, (S,R)-isomer ofactinol, was analyzed to be 0.7 g/l. TABLE 1 Ketoisophorone-reducingreaction using S. cerevisiae INVScI cells time titer (g/l) (hr)ketoisophorone (R) -levodione (R, R) -actinol (S, R) -actinol 0 3.600.00 0.00 0.00 1 3.69 0.00 0.00 0.02 2 2.65 0.53 0.00 0.05 4 2.25 1.350.00 0.12 6 1.06 1.94 0.01 0.18 17 0.00 2.78 0.07 0.65 19 0.00 2.58 0.070.60

EXAMPLE 2

Introduction of the Levodione Reductase Gene Derived From C. aquaticumAKU611 Into S. cerevisiae INVScI

A DNA fragment encoding levodione reductase gene has already been clonedby using PCR primers designed from the information based on partialamino acid sequences of purified levodione reductase as described below.

At first, genomic DNA of Corynebacterium aquaticum AKU611 (FERM BP-6448)was prepared using Genome Isolation Kit (BIO101). Using the preparedgenomic DNA as template, a complete coding sequence for the levodionereductase gene without excessive flanking region was obtained by PCRamplification using a thermal cycler (Perkin elmer 2400, U.S.A.). Thetwo synthetic primers used were: LV-ORF(+)(SEQ ID NO:1) (having an EcoRIsite GAATTC) and LV-ORF(−) (SEQ ID NO:2) (having an PstI site CTGCAG).The PCR mixture (0.02 ml) contained 5 pmol of each primer, 0.2 mM ofeach dNTP, and 1 U of LA Taq (Takara Shuzo co.LTD/Kyoto, Japan). Theinitial template denaturation step consisted of 1 min at 94° C. Anamplification cycle of 20 sec at 98° C., 2 min at 70° C. and 4 min at72° C. was repeated for 25 times.

By this reaction, a DNA fragment containing a complete ORF of thelevodione reductase gene (0.8 Kb) was amplified. This amplifiedlevodione reductase gene was treated with EcoRI and PstI, and ligatedwith a vector, pKIC223-3 (Amersham Bioscience I Buckinghamshire,England) that was predigested with EcoRI and PstI to construct aplasmid, pKKLR(1-15). E. coli JM109 was transformed with the ligationmixture, and several clones were selected for sequence analysis. Thesequence of the cloned levodione reductase gene of each candidate clonewas examined. One of the clones that showed completely the same sequenceas the levodione reductase sequence of Corynebacterium aquaticum AKU611(FERM BP-6448) was named as JM109[pKKLR(1-15)]. pKKLR(l-15) was isolatedfrom this strain and used for further experiments.

The 0.8 kb DNA fragment encoding levodione reductase gene was cut outfrom plasmid pKKLR(1-15) which is comprising levodione reductase geneand E. coli vector pKK223-3, and inserted into pYES2, which is anexpression vector for S. cerevisiae. GAL1 promoter and CYC1 terminator,both of which are originally existing on pYES2, are connected to thelevodione reductase gene on the plasmid as a result of ligation of the0.8 kb levodione reductase fragment with EcoRI-digested form of theplasmid pYES2, followed by blunting of the ligated DNA and self-ligationof the DNA. After confirming the direction of the inserted levodionereductase gene, this plasmid, named as pLVRS1, was used to transform S.cerevisiae INVScI. The plasmid is illustrated in the Figure, depictingplasmid DNA, pLVRS1, to introduce LVR gene into Z. bailii ATCC11486cells. P_(GAL1), T_(CYC1), pMB1 ori, AmP^(R), URA3, 2 micron ori and flori means GAL1 promoter, CYC1 terminator, pMB1 (pUC-derived plasmid)origin, ampicillin resistance gene, URA3 gene, 2 micron DNA origin andfl origin, respectively. LVR gene is described by gray arrow. Thelevodione reductase-encoding plasmid pLVRS1 was introduced into S.cerevisiae INVScI cells by using S. c. EasyComp Transformation Kit(Invitrogen). The method of the transformation was basically the same asthe protocol of the transformation kit. Transformed cells can beselected by spreading resulting transformation solution onto solidsynthetic medium which lacks uracil because of the presence of theuracil auxotroph of the parent strain and URA3 marker of the plasmid,and incubating these plates for 4 days at 30° C. Presence of the plasmidin the yeast strain was confirmed by checking occurrence ofpLVRS1-containing E. coli DH5 alpha strains after transformation of theE. coli strain by using total DNA of the candidate of S. cerevisiaeINVScI transformant strain as a donor DNA. The strain possessing PLVRS1,S. cerevisiae (pLVRS1), was then used for further experiments.

EXAMPLE 3

Production of Actinol By Reducing Ketoisophorone Using S. cerevisiaeINVScI Resting Cells Containing the Levodione Reductase Gene DerivedFrom C. aquaticum AKU611

The ketoisophorone-reducing reaction using resting cells of S.cerevisiae INVScI possessing pLVRS1 was originally performed bybasically the same method as described in Example 1. The result of thereaction is described in Table 2. After 19.0 hr reaction, 1.6 g/l ofactinol was produced from 5.0 g/l of ketoisophorone. Optical purity foractinol was not so high (67.2%) because of the relatively high value of(S,R)-isomer concentration (0.48 g/l). TABLE 2 Ketoisophorone-reducingreaction using S. cerevisiae INVScI possessing pLVRS1 Time titer (g/l)optical purity for (hr) KIP (R)-LDN (R, R)-ACT (S, R)-ACT (S)-PHO (R,R)-ACT (% e.e) 0 3.79 0.00 0.00 0.00 0.00 1 3.02 0.00 0.03 0.01 0.3366.6 2 2.44 0.39 0.12 0.03 0.52 73.0 4 1.61 0.83 0.31 0.07 0.62 77.8 61.00 1.12 0.55 0.12 0.60 78.8 17 0.11 1.24 1.31 0.36 0.29 71.0 19 0.061.72 1.60 0.48 0.27 67.2KIP: ketoisophorone;(R)-LDN: (R) -levodione;(R, R)-ACT: (R, R)-actinol;(S, R)-ACT: (S, R)-actinol,(S)-PHO: (S)-phorenol

0.33 g of resting cells were suspended into 1 ml of reaction buffer,which was 0.1 M of KPB (pH 6.0) containing 150 g/l of glucose and 10.0g/l of ketoisophorone, and incubated at 20° C. in a test tube withgently shaking (200 rpm). The reaction solution was recovered,extracted, and analyzed by gas chromatography as described in Example 1.

The result of the reaction is described in Table 3. After 25.0 hrreaction, 3.3 g/l of actinol was produced from 10.0 g/l ofketoisophorone. Since the concentration of (S,R)-isomer could besuppressed to 0.52 g/l, optical purity for actinol was increased to81.5%. TABLE 3 Ketoisophorone-reducing reaction using S. cerevisiaeINVScI possessing pLVRS1 Time titer (g/l) optical purity for (hr) KIP(R)-LDN (R, R)-ACT (S, R)-ACT (S)-PHO (R, R)-ACT (% e.e) 0 11.23 0.000.00 0.00 0.00 1.5 6.78 0.00 0.09 0.00 0.99 100.0 3.0 5.93 0.57 0.350.06 1.88 81.6 4.5 3.86 0.86 0.65 0.09 2.10 85.9 6.0 2.80 0.99 0.94 0.122.18 85.2 7.5 2.27 1.20 1.14 0.14 2.08 85.3 9.0 1.77 1.23 1.34 0.16 2.0685.4 22.0 0.35 1.63 3.20 0.47 1.27 82.6 23.5 0.31 1.64 3.29 0.50 1.1881.9 25.0 0.25 1.51 3.34 0.52 1.08 81.5 26.5 0.21 1.42 3.33 0.56 0.9980.3KIP: ketoisophorone;(R)-LDN: (R) -levodione;(R, R)-ACT: (R, R)-actinol;(S, R)-ACT: (S, R)-actinol,(S)-PHO: (S)-phorenol

1. A process for producing actinol from ketoisophorone which comprisescontacting ketoisophorone with a recombinant microorganism or cell-freeextract thereof in a reaction mixture, wherein said recombinantmicroorganism is obtainable by transforming a host microorganism, e.g.selected from the group consisting of microorganisms of the generaSaccharomyces, Zygosaccharomyces, and Candida, such as commerciallyavailable baker's yeast, Saccharomyces cerevisiae ATCC7754,Saccharomyces rouxii (Zygosaccharomyces rouxii) HUT7191 (IPO 0494),Saccharomyces delbrueckii HUT7116 (Saccharomyces unisporus IFO 0298),Saccharomyces delbrueckii (Torulaspora delbrueckii) HUT7102,Saccharomyces willianus HUT7106, Zygosaccharomyces bailii ATCC11486,Candida tropicalis IFO 1403, and a mutant thereof, which is capable ofreducing ketoisophorone to levodione with a levodione reductase gene,e.g. a levodione reductase gene derived from a microorganism belongingto the genus Corynebacterium, such as C. aquaticum AKU611 (FERM BP-6448)or a mutant thereof, and isolating the produced actinol from thereaction mixture.
 2. The process according to claim 1, wherein thereaction is carried out at pH values of from 4.0 to 9.0, preferably from5.0 to 8.0, and at a temperature range from 10 to 50° C., preferablyfrom 20 to 40° C., and for 15 minutes to 72 hours, preferably for 30minutes to 48 hours.
 3. A process for producing actinol fromketoisophorone which comprises contacting ketoisophorone with arecombinant microorganism or cell-free extract thereof in a reactionmixture, wherein said recombinant microorganism is obtainable bytransforming a host microorganism, e.g. selected from the groupconsisting of microorganisms of the genera Cellulomonas,Corynebacterium, Planococcus, and Arthrobacter, such as Cellulomonas sp.AKU672 (FERM BP-6449), Corynebacterium aquaticum AKU610 (FERM BP-6447),C. aquaticum AKU611 (FERM BP-6448), P. okeanokoites AKU152 (IFO 15880),A. sulfureus AKU635 (IFO 12678), and a mutant thereof, which is capableof reducing levodione to actinol with a ketoisophorone reductase gene,e.g. derived from a microorganism belonging to the genera Saccharomyces,Zygosaccharomyces, or Candida, such as commercially available baker'syeast, S. cerevisiae ATCC7754, S. rouxii (Z. rouxii) HUT7191 (IFO 0494),S. delbrueckii HUT7116 (S. unisporus IFO 0298), S. delbrueckii(Torulaspora delbrueckii) HUT7102, S. willianus HUT7106, Z. bailiiATCC11486, C. tropicalis IFO 1403, and a mutant thereof, and isolatingthe resulted actinol from the reaction mixture.
 4. The process accordingto claim 3, wherein the reaction is carried out at pH values of from 4.0to 9.0, preferably from 5 to 8.0, and at a temperature range from 10 to50° C., preferably from 20 to 40° C., and for 15 minutes to 72 hours,preferably for 30 minutes to 48 hours.
 5. A process for producingactinol from ketoisophorone which comprises contacting ketoisophoronewith a recombinant microorganism or cell-free extract thereof in areaction mixture, wherein said recombinant microorganism, e.g. selectedfrom the group consisting of microorganisms of the genera Cellulomonas,Corynebacterium, Planococcus, and Arthrobacter, such as Cellulomonas sp.AKU672 (FERM BP-6449), Corynebacterium aquaticum AKU610 (FERM BP-6447),C. aquaticum AKU611 (FERM BP-6448), P. okeanokoites AKU152 (IFO 15880),A. sulfureus AKU635 (IFO 12678), and a mutant thereof, expresses bothketoisophorone reductase gene and levodione reductase gene, e.g. alevodione reductase gene derived from a microorganism belonging to thegenus Corynebacterium, such as C. aquaticum AKU611 (FERM BP-6448) or amutant thereof, and isolating the produced actinol from the reactionmixture.
 6. The process according to claim 5, wherein the reaction iscarried out at pH values of from 4.0 to 9.0, preferably from 5.0 to 8.0,and at a temperature in the range of from 10 to 50° C., preferably from20 to 40° C., and for 15 minutes to 72 hours, preferably for 30 minutesto 48 hours.
 7. A process for producing actinol from ketoisophorone bycontacting ketoisophorone with purified ketoisophorone reductase, e.g.derived from a microorganism belonging to the genera Saccharomyces,Zygosaccharomyces, or Candida, such as commercially available baker'syeast, S. cerevisiae ATCC7754, S. rouxii (Z. rouxii) HUT7191 (IFO 0494),S. delbrueckii HUT7116 (S. unisporus IFO 0298), S. delbrueckii(Torulaspora delbrueckii) HUT7102, S. willianus HUT7106, Z. bailiiATCC11486, C. tropicalis IFO 1403, and a mutant thereof, which iscapable of catalyzing the conversion of ketoisophorone to levodione andpurified levodione reductase, e.g. a levodione reductase derived from amicroorganism belonging to the genus Corynebacterium, such as C.aquaticum AKU611 (FERM BP-6448) or a mutant thereof, which is capable ofcatalyzing the conversion of levodione to actinol simultaneously.
 8. Theprocess according to claim 7, wherein the reaction is carried out at pHvalues of from 4.0 to 9.0, preferably from 5.0 to 8.0, at a temperaturein the range of from 10 to 50° C., preferably from 20 to 40° C., and for5 minutes to 48 hours, preferably for 15 minutes to 24 hours.
 9. Arecombinant microorganism that is obtainable by transforming a hostorganism, e.g. a microorganism belonging to the genera Saccharomyces,Zygosaccharomyces, or Candida, such as commercially available baker'syeast, S. cerevisiae ATCC7754, S. rouxii (Z. rouxii) HUT7191 (IFO 0494),S. delbrueckii HUT7116 (S. unisporus IFO 0298), S. delbrueckii(Torulaspora delbrueckii) HUT7102, S. willianus HUT7106, Z. bailiiATCC11486, C. tropicalis IFO 1403, and a mutant thereof, which iscapable of reducing ketoisophorone to levodione with a levodionereductase gene, e.g. a levodione reductase gene derived from amicroorganism belonging to the genus Corynebacterium, such as C.aquaticum AKU611 (FERM BP-6448) or a mutant thereof.
 10. A recombinantmicroorganism that is obtainable by transforming a host organism, e.g. amicroorganism of the genera Cellulomonas, Corynebacterium, Planococcus,and Arthrobacter, such as Cellulomonas sp. AKU672 (FERM BP-6449),Corynebacterium aquaticum AKU610 (FERM BP-6447), C. aquaticum AKU611(FERM BP-6448), P. okeanokoites AKU152 (IFO 15880), A. sulfureus AKU635(IFO 12678), and a mutant thereof, which is capable of reducinglevodione to actinol with ketoisophorone reductase gene, e.g, derivedfrom a microorganism belonging to the genera Saccharomyces,Zygosaccharomyces, or Candida, such as commercially available baker'syeast, S. cerevisiae ATCC7754, S. rouxii (Z. rouxii) HUT7191 (IFO 0494),S. delbrueckii HUT7116 (S. unisporus IFO 0298), S. delbrueckii(Torulaspora delbrueckii) HUT7102, S. willianus HUT7106, Z. bailiiATCC11486, C. tropicalis IFO 1403, and a mutant thereof.
 11. Arecombinant microorganism which expresses both ketoisophorone reductasegene, e.g, derived from a microorganism belonging to the generaSaccharomyces, Zygosaccharomyces, or Candida, such as commerciallyavailable baker's yeast, S. cerevisiae ATCC7754, S. rouxii (Z. rouxii)HUT7191 (IFO 0494), S. delbrueckii HUT7116 (S. unisporus IFO 0298), S.delbrueckii (Torulaspora delbrueckii) HUT7102, S. willianus HUT7106, Z.bailii ATCC11486, C. tropicalis IFO 1403, and a mutant thereof, and alevodione reductase gene, e.g. a levodione reductase gene derived from amicroorganism belonging to the genus Corynebacterium, such as C.aquaticum AKU611 (FERM BP-6448) or a mutant thereof.